The present invention is directed to N-Acetyl-D-Glucosamine and methods of making N-Acetyl-D-Glucosamine.
Chitin is a natural polysaccharide present in various marine and terrestrial organisms, including crustacea, insects, mollusks, and microorganisms, such as fungi. The structure of chitin is that of an unbranched polymer of 2-acetoamido-2-deoxy-D-glucose (also known as poly (N-acetyl-D-glucosamine)), and can be represented by the general repeating structure: 
Chitin is typically an amorphous solid that is largely insoluble in water, dilute acids, and alkali. Although chitin has many uses, it can also be degraded to form other useful materials, such as carbohydrates, one of which is the amino sugar N-acetyl-D-glucosamine (NAG). N-acetyl-D-glucosamine typically includes a single glucosamine unit, but can also include small amounts of short oligomers, such as chitobiose or chitotriose, that have two and three glucosamine units, respectively. N-acetyl-D-glucosamine can be used for various applications, including as a food additive and in pharmaceutical compositions.
The most common source of chitin for use in making N-acetyl-D-glucosamine is shellfish (such as shrimp) biomass. Unfortunately, limitations exist with the recovery of N-acetyl-D-glucosamine from shellfish biomass. One problem with recovering N-acetyl-D-glucosamine from shellfish is that it is very difficult to obtain uniform shellfish biomass. The uniformity problems occur in part because shellfish often vary by size, age, and species; are grown under varied environmental conditions; and are gathered from diverse locations. This lack of uniformity makes it difficult to precisely process shellfish biomass. In addition, some quality control issues can arise due to the fact that N-acetyl-D-glucosamine obtained from crustacea can have a high ash content and can contain heavy metals that are concentrated in the crustacea from their aquatic environment. A further problem with N-acetyl-D-glucosamine derived from harvested crustacea is that it has the potential to include undesired proteins and allergens.
Therefore, a need exists for an improved N-acetyl-D-glucosamine material that is obtained utilizing a non-shellfish chitin source.
The present invention is directed to N-acetyl-D-glucosamine obtained from microbial biomass, and to methods of obtaining N-acetyl-D-glucosamine from microbial biomass. In particular, the present invention is directed to the use of fungal biomass to obtain N-acetyl-D-glucosamine. The N-acetyl-D-glucosamine is efficiently obtained at high purity by degrading chitin in the biomass to create N-acetyl-D-glucosamine.
The fungal biomass generally contains a significant amount of glucan intermixed with the chitin. Glucan is a high molecular weight polymer of glucose and is derived from the cell wall of microbial biomass. Glucan can include linkages that are exclusively beta linkages, exclusively alpha linkages, or a mixture of alpha and beta linkages. The glucan components of the fungal biomass bind and immobilize the chitin materials, and generally make the chitin unavailable for efficient degradation into N-acetyl-D-glucosamine. The present invention overcomes the problems associated with the presence of glucan components by degrading the glucan sufficiently to gain chemical access to the chitin components, which are subsequently degraded to form N-acetyl-D-glucosamine.
The methods of recovering N-acetyl-D-glucosamine generally include providing fungal biomass containing chitin and glucan; degrading at least a portion of the glucan; and degrading at least a portion of the chitin to produce N-acetyl-D-glucosamine. In one implementation of the invention the chitin and glucan are enzymatically degraded, while in other implementations the chitin and glucan are chemically degraded.
The biomass used to form the N-acetyl-D-glucosamine of the invention typically is a fungal biomass that has at least 5 weight percent chitin and at least 5 weight percent glucan on a dry basis before degradation, and even more typically has at least 15 weight percent chitin and 50 weight percent glucan on a dry basis before degradation.
Various fungal biomass sources can be used, including fungal biomass derived from Aspergillus sp., Penicillium sp., Absidium sp., Lactarius sp., Mucor sp., Saccharomyces sp., Candida sp. or combinations thereof. As used herein, xe2x80x9csp.xe2x80x9d refers to either singular xe2x80x9cspeciexe2x80x9d or plural xe2x80x9cspeciesxe2x80x9d. Suitable specific microbial biomasses include, without limitation, Aspergillus niger, Aspergillus terreus, Aspergillus oryzae, Lactarius vellereus, Mucor rouxii, Penicillium chrysogenum, Penicillium notatum, Saccharomyces cerevisiae; and in particular, Candida guillermondi, Aspergillus niger, and Aspergillus terreus. Generally the biomass is recovered from a commercial fermentation reaction, including the commercial production of organic acids, such as citric acid and itaconic acid. As use herein, the term microbial when used to describe the fungal biomass source does not include phyto-plankton and crustaceans or mollusks, which differ significantly in composition and properties from fungal biomass.
In one implementation of the invention the chitin and glucan components of the fungal biomass are degraded by enzymes. The enzymes are typically microorganism-derived. The enzymes can be added to the fungal biomass in the presence of the microorganisms from which they are derived, which has the advantage of avoiding the step of separating the enzymes from their source organisms. Inclusion of the source microorganisms can also be advantageous in implementations where the microorganisms continue to create enzymes after having been added to the fungal biomass. In other implementations the enzymes are separated from their source microorganisms (such as by filtration or centrifugation) and subsequently added to the fungal biomass.
Suitable enzymes for degrading the chitin and glucan include chitinases, xcex2-N-acetyl-glucosaminidases, and glucanases. The enzymes are generally secreted from eucaryotic or prokaryotic microorganisms. Suitable eucaryotic organisms include those from the Trichoderma genus, including, for example, Trichoderma harizianum and Trichoderma reesei. Suitable prokaryotic organisms include those from the Serratia genus, the Streptomyces genus, and the Nocardia genus. Example prokaryotic organisms include Serratia marcesens, Streptomyces griseus, and Nocardia orientalis. Glucanase enzymes are particularly advantageous in order to degrade the glucan component of the fungal biomass. These glucanases should be present at relatively high concentrations in typical implementations of the invention.
In general it is not necessary to subject the fungal biomass to extensive physical or chemical pretreatments prior to processing to create N-acetyl-D-glucosamine. Thus, it is usually not necessary to grind the fungal biomass or to pretreat it with an organic solvent. Such steps are normally not required, but they can optionally be performed in some implementations of the invention. When enzymes are used to degrade the chitin and glucan, the degradation reaction is usually maintained at a pH of 4.0 to 6.0 and a temperature of 20xc2x0 to 45xc2x0 C. Higher temperatures are generally advantageous, but the temperatures must be low enough to keep the enzymes from degrading.
Despite the high levels of glucan present in the fungal biomass, the resulting N-acetyl-D-glucosamine is generally of high purity. Typically the resulting N-acetyl-D-glucosamine makes up at least 85 percent of the chitin-derived carbohydrates from the fungal biomass.
The above summary of the present invention is not intended to describe each disclosed embodiment of the present invention. This is the purpose of the detailed description and claims that follow.